REVIEW Open Access

Advances in therapeutic use of a drug-stimulated translational readthrough ofpremature termination codonsMaciej Dabrowski, Zuzanna Bukowy-Bieryllo and Ewa Zietkiewicz*

Abstract

Premature termination codons (PTCs) in the coding regions of mRNA lead to the incorrect termination of translationand generation of non-functional, truncated proteins. Translational readthrough of PTCs induced by pharmaceuticalcompounds is a promising way of restoring functional protein expression and reducing disease symptoms, withoutaffecting the genome or transcriptome of the patient. While in some cases proven effective, the clinical use ofreadthrough-inducing compounds is still associated with many risks and difficulties. This review focuses on problemsdirectly associated with compounds used to stimulate PTC readthrough, such as their interactions with the cell andorganism, their toxicity and bioavailability (cell permeability; tissue deposition etc.). Various strategies designed toovercome these problems are presented.

Keywords: Translational readthrough, Stop codon suppression, Nonsense suppression, Premature termination codon,Genetic diseases

BackgroundMost of the reviews on the premature termination co-dons (PTCs) readthrough studies focus on the efficiencyof the PTC suppression-stimulating agents, without ex-ploring the associated problems of these agents’ toxicityand bioavailability. The present review, by providing adifferent perspective, aims to contribute to understand-ing of the problems associated with using PTC read-through strategies. The risks and difficulties associatedwith compounds used to stimulate PTC readthrough,mostly related with their interaction with the cell and or-ganism, and concerning their toxicity and bioavailability(cell permeability; tissue deposition etc.) are discussed. Inaddition, strategies to overcome these problems are dem-onstrated. We also present outcomes of recently finishedclinical trials on the readthrough compounds. The aspectof PTC identity and its nucleotide context has been re-cently reviewed (Dabrowski et al., 2015) and is not exten-sively discussed here; other problems, related to themolecular side of PTC-RT, deserve a separate review andare only briefly mentioned.

Main textPremature termination codonsAn extensive meta-analysis study, based on the HumanGene Mutation Database, has revealed that 12% of alldescribed gene lesions causing human inherited diseasesis caused by nonsense mutations (Mort et al., 2008). Thesemutations, by changing an amino acid coding triplet intoa stop codon, introduce premature termination codons,PTCs, into the protein-coding gene sequence. PTCs mayalso be caused by other types of mutations, such as frame-shifts (insertion or deletion other than multiple-of-threebase pairs) or mutations in the conserved splice-site se-quences (leading to a defective intron removal from thepre-mRNA) (Mort et al., 2008; Mendell & Dietz, 2001).PTCs in mRNAs can even occur independently of anychanges in DNA, through the aberrant mRNA processing(alternative splicing) (Pan et al., 2006; Lewis et al., 2003).In any case, PTCs located in the coding regions of

mRNA lead to the premature termination of translationand, as a consequence, to the production of truncatedproteins (Mendell & Dietz, 2001). In most cases, this re-duces the amount of a full-length protein in a recessive-negative manner, whereby mutations of both alleles arenecessary to result in a deleterious phenotype (Khajavi et* Correspondence: ewa.zietkiewicz@igcz.poznan.pl

Institute of Human Genetics; Polish Academy of Sciences, Poznan, Poland

Molecular Medicine

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Dabrowski et al. Molecular Medicine (2018) 24:25 https://doi.org/10.1186/s10020-018-0024-7

al., 2006). However, PTCs can also exert dominant-negative effect (Inoue et al., 2004). Phenotypic manifest-ation usually involves loss-of-function effects, but thereare examples of gain-of-function events (Manuvakhova etal., 2000). For example, in β-thalassemia, truncated pro-teins derived from PTC-bearing transcripts are respon-sible for generation of insoluble globin chains, which aretoxic to the cell (Thein et al., 1990).The synthesis of deleterious C-terminally truncated

proteins in eukaryotic organisms is reduced due to thespecialized mRNA surveillance mechanisms. One of themis the nonsense-mediated mRNA decay (NMD) pathway(Lejeune, 2017a). This conserved quality-control processdetects and degrades transcripts containing abnormalitiessuch as: PTCs, introns downstream of the terminationcodon, long 3′ untranslated regions (3′UTRs) or upstreamopen reading frames (uORFs) (Mühlemann, 2008; Hogg &Goff, 2010; Miller & Pearce, 2014; Lejeune, 2017b).Many efforts have been made to develop therapeutic

strategies that would counteract negative effects of PTCs.One of them relies on exploring the natural phenomenonof termination codon suppression through the transla-tional readthrough mechanism.

Basal translational readthroughEffective termination of protein translation in eukaryotesrequires recognition of the stop codon, localized in the A-site of the ribosome, by a release factor, eRF1. The eRF1further interacts with another release factor – eRF3, andwith GTP. This ternary termination complex (eRF1-eRF3-GTP) interacts with the poly(A)-binding protein (PABP)present at the 3′-UTR of mRNA; this leads to efficient hy-drolysis of GTP and cleavage of the peptidyl-tRNA bond(Bulygin et al., 2017). As a consequence, the newly synthe-sized polypeptide chain is released from the ribosome.With the termination of translation being based on the

steric match between the stop codon and the eRF1, thisprocess is not 100% efficient. Stop codons in mRNA canbe suppressed (recoded) through the natural mechanismof basal translational readthrough (Dabrowski et al., 2015;Fearon et al., 1994). In this process, a near-cognate tRNA(nc-tRNA) outcompetes eRF1 at the A-site of the ribo-some. Nc-tRNAs have anticodons, which are complemen-tary to only two of the three positions of a nonsensecodon in mRNA. The recent studies indicate, that theinteraction between PTC and a nc-tRNA anticodon oc-curs by mispairing at either position 3 or 1 of the stopcodon (Roy et al., 2015; Roy et al., 2016). If the nc-tRNAinteracts with the PTC at the A-site, the amino acidtransported by such nc-tRNA is incorporated into thesynthesized polypeptide chain, and translation – insteadof being terminated – continues until the next in-framestop codon (Fig. 1).

Not all stop codons can be read through with the sameefficacy. The level of “leakiness” of the stop codons can beranked as UGA>UAG>UAA (Manuvakhova et al., 2000).Also the sequence of nucleotides upstream and down-stream to the stop codon has the effect on the efficacy ofthe readthrough process. The majority of evidence indicatesthat a nucleotide immediately following the terminationcodon in the 3′ direction (position + 4, when the first nu-cleotide of the stop codon is marked as + 1), is in-volved in the interactions between mRNA and translationalmachinery (McCaughan et al., 1995; Jungreis et al., 2011;Loughran et al., 2014). This suggests that the actualtranslation termination signal consists of a tetranu-cleotide sequence, rather than only the stop codon itself(Brown et al., 1990). Recently, it has been proposed, basedon genetic interaction studies in yeast, that + 4 cytosinecompromises the ability of eRF1 to recognize appropri-ately a stop codon, thus providing precedence for the im-portance of nucleotide positioning for eRF1-mediatedtermination efficiency (Beznosková et al., 2016). In addition,the upstream sequence, immediately 5′ of the stop codon(positions − 1 to − 3) also exerts an effect; this effect is con-sidered to be more subtle than the downstream sequencecontext of the transcript. Studies suggest that even relativelydistant nucleotides (positions + 5, + 6, + 9) may also influencethe translational readthrough (for a review, see Dabrowski etal., 2015, RNA Biology) (Dabrowski et al., 2015).It has been estimated that basal readthrough of normal

termination codons (NTCs) occurs in 0.001 to 0.1% oftotal rounds of translation of a given transcript (Keelinget al., 2012). In the case of PTCs, basal readthroughlevels are higher and can range from 0.01% to even 1%(Manuvakhova et al., 2000; Bonetti et al., 1995; Cassan &Rousset, 2001). It is not known, why the basal read-through levels differ so much. It has been hypothesized,that efficient translation termination requires the closecontact between the release factors interacting withNTC and PABPs (Lejeune, 2017a; Cosson et al., 2002;Celik et al., 2015). Presence of a PTC in the transcript,usually more distant from the 3’UTR, could limit theinteraction between eRFs and PABPs, leading to the lessefficient action of eRF3 and the delayed release of thetranscript from the ribosome (Ivanov et al., 2008). Theprolonged presence of the translational machinery at thePTC would increase the susceptibility to the basal transla-tional readthrough of the PTC (PTC-RT) (Amrani et al.,2004).

Drug-induced translational readthroughA number of studies have demonstrated that certainlow-molecular-mass drugs can stimulate recoding of aPTC by the translation machinery (Lee & Dougherty,2012). These findings have opened the way to new thera-peutic approaches to nonsense mutations in genes

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associated with genetic diseases. However, the efficientuse of this approach in the clinic is still hampered by anumber of problems (Fig. 2).Aminoglycoside antibiotics (AAGs) are among the

most studied drugs capable of inducing PTC-RT -

already in 1960s, first papers indicating readthroughpotential of AAGs in bacteria have been published(Davies et al., 1964; Anderson et al., 1965). In 1985, thisprocess was demonstrated in mammalian cells (Burke &Mogg, 1985). AAGs are oligosaccharides consisting of

Fig. 2 Different problems associated with the drug-stimulated PTC-RT therapy. Upper part – problems associated with the PTC-RT- stimulating drugs.Lower part – problems associated with the biology of PTC-RT process

Fig. 1 PTC-RT process. a Ribosome encounters premature termination codon (PTC); the site is recognized by the translation termination machineryand the polypeptide elongation is prematurely terminated. b After addition of readthrough compound, translational machinery decodes PTC (PTC isrecognized by nc- tRNAs), and translation continues until the normal termination codon (NTC). It allows translating a full-length protein. The NMDsurveillance mechanism may detect and degrade PTC-bearing transcripts. If the NMD process is inhibited, very low levels of the full-length protein canbe present even in the absence of stimulating agents, as the result of the endogeneous suppression of PTC

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streptidine or 2-deoxystreptidine as the molecular core,with a variable number of sugar rings and ammoniumgroups (François et al., 2005). Commonly used to treatGram-negative bacterial infections, they bind with theseven-nucleotide loop structure in the decoding center ofthe bacterial ribosome and interfere with its function(Fan-Minogue & Bedwell, 2008). This enables effectivemisincorporation of the nc-tRNAs, resulting in extensivetranslational misreading, followed by a complete inhib-ition of protein synthesis in bacteria. In eukaryotes, asmall difference in the rRNA nucleotide sequence signifi-cantly lowers the efficiency of this interaction (Lynch &Puglisi, 2001). Nonetheless, in the case of PTC, the impactof AAGs on the translational apparatus is often sufficientto reduce discrimination between cognate and nc-tRNAs,and to enhance translational readthrough.The therapeutic potential of AAGs as PTC readthrough-

stimulating compounds has been tested in many models,from the in vitro transcription and translation system(Manuvakhova et al., 2000; Du et al., 2002; Keeling &Bedwell, 2002; Lai et al., 2004), through dual reporters incell lines (Howard et al., 2004; Bidou et al., 2004; Floquet etal., 2011; Bukowy-Bieryllo et al., 2016), patient derived cells(Salvatori et al., 2009; Nakamura et al., 2012; Cogan et al.,2014; Dündar et al., 2017). The first in vivo demonstrationof the therapeutic AAG properties has been in mdx mice,an animal model of Duchenne muscular dystrophy (DMD),where gentamicin treatment restored 10–20% of normaldystrophin levels in the skeletal muscles (Barton-Davis etal., 1999). Since then, a large variety of AAGs has beentested for their PTC-RT-stimulating properties, both in ani-mal models (Arakawa et al., 2003; Du et al., 2006; Guerin etal., 2008; Yukihara et al., 2011; Rowe et al., 2011; Gunn etal., 2014) and clinical trials (Clancy et al., 2001; Wilschanskiet al., 2003; Politano et al., 2003; Sermet-Gaudelus et al.,2007; Malik et al., 2010). The latest pilot clinical study(ClinicalTrials.gov; id: NCT02698735) has shown positiveresults of gentamicin use in recessive dystrophic epidermo-lysis bullosa patients with nonsense mutation in the geneencoding type VII collagen. Topical or intradermal adminis-tration of gentamicin increased the level of full-length colla-gen VII from 20 to 165% of the wild-type level, theexpression of the protein persisted for 3 months. Inaddition, a significant reduction of disease symptoms wasobserved (improved epidermal-dermal adherence, reducedblister formation, and enhanced wound closure) (Woodleyet al., 2017).

Toxicity of AAGsEven though AAGs have been commonly used in clinicsfor decades, they may exert strong oto- and nephrotoxiceffects on the organism. Accumulation of AAGs in therenal epithelial cells leads to apoptosis and necrosis ofthese cells (Lopez-Novoa et al., 2011). However, this

effect is reversible after stopping AAGs administration(Mingeot-Leclercq et al., 1999). The negative influenceof AAGs on the renal function in patients can be also di-minished by the appropriate hydration therapy and ap-plication of dialysis, when necessary (Lopez-Novoa et al.,2011). The ototoxicity induced by AAGs manifests as ir-reversible, symmetric, bilateral sensorineural hearingloss. Due to the very slow removal of AAGs from theinner ear fluids, the onset of hearing loss can occur days,or even weeks, after finishing the AAG administration(Huth et al., 2011). Inside the hair cell, AAGs causedamage, either directly or indirectly, first by causing dis-array of stereocilia, and ultimately by activating apop-tosis (Abi-Hachem et al., 2010).The selective toxicity observed in the inner ear and

kidneys is associated with the cellular uptake of AAGsthrough megalin, an endocytic receptor localized in the ap-ical membrane of epithelial cells present in the proximalrenal tubules and in the hair cells of the inner ear(Moestrup et al., 1995). The mechanism, by which AAGsinduce apoptosis, both in the renal cells and in the haircells, is to date not well-defined. Some evidence indicates arole of reactive oxygen species (ROS) (Huth et al., 2011).Positively-charged AAGs are able to interact with variousnegatively-charged cellular components such as phospho-lipids, phospholipases, and metal ions. AAGs bounded tophospholipids form complexes, which can aggregate in theinternal lysosomal membranes, causing phospholipidosis, acondition often associated with nephro- and ototoxicity(Couture et al., 1994; Reasor & Kacew, 2001). More import-antly, the AAG-phospholipid interaction leads to the gener-ation of ROS, which affect membrane fluidity andpermeability, disrupt the activity of enzymes, ion chan-nels and receptors, and finally direct the cell to theapoptotic pathway (Xie et al., 2011). Another explanationfor the cell apoptosis related to AAGs administration sug-gests involvement of the decoding site of the ribosome ofmitochondrial ribosomes, which closely resembles theA-site of bacterial ribosomes. The interaction ofAAGs with the 12S rRNA at the A-site causes mis-translation, then inhibition of protein synthesis, ultimatelyleading to cell death (Hobbie et al., 2008). AAG-mediatedoxidative damage of the mitochondrial enzyme – aconitasecan also cause an increase in superoxide overproductionfrom the free ferrous iron in mitochondria, that in turnincreases the amount of hydroxyl radicals via the Fentonreaction, and finally induces the cell apoptosis (Shulman etal., 2014).In spite of these adverse effects, one study has demon-

strated that AAGs used to stimulate PTC-RT can berelatively safe. The six month-long gentamicin therapyin DMD patients was completed safely, with no impair-ment of either renal or hearing function. The negativeeffects of AAGs were avoided thanks to the informed

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choice of the study cohort and a careful study design; allsubjects with the A1555G mutation in 12S rRNA geneof mtDNA, known to predispose to gentamicin-inducedototoxicity, had been excluded from the treatment, whilein the remaining patients a strict regimen of gentamicinadministration was obeyed (Malik et al., 2010).Although the clinical use of AAGs for a prolonged

time is associated with many risks, the benefits of a pos-sible PTC-RT-based therapy are indisputable. To allevi-ate the problems resulting from AAGs toxicity, variousattempts have been undertaken to mitigate the toxicityor to select other compounds with PTC-RT-inducingpotential but without undesirable side effects of AAGs.

Overcoming cytotoxic effects of AAGsOne approach tested for reducing the cytotoxicity ofAAGs involves their co-administration with differentcompounds, which are able to reduce interaction ofAAGs with different cellular targets. This strategy wasshown to be successful for tobramycin or gentamicin co-administrated with a lipopeptic antibiotic, daptomycin(Beauchamp et al., 1990; Thibault et al., 1994). Nega-tively charged daptomycin complexed AAGs through anelectrostatic interaction, preventing them from bindingto phospholipids and other intracellular targets (Coutureet al., 1994). Co-administration of gentamicin with an-other compound, the negatively charged poly-L-asparticacid (PAA), was shown to protect rats against the develop-ment of kidney nephrotoxicity (Gilbert et al., 1989;Ramsammy et al., 1989). It has been suggested that PAA,similarly to daptomycin, binds AAGs and prevents phos-pholipidosis, which would otherwise lead to nephrotox-icity (Kishore et al., 1990). In addition, PAA increased theintracellular concentration of gentamicin, contributing toa higher level of PTC-RT. It also slowed-down eliminationof AAGs from cytosol, extending the time period duringwhich readthrough stimulation occurred. Similar resultswere obtained in the mouse model of cystic fibrosis(Du et al., 2009a).Another approach used to mitigate the toxic effects of

AAGs relies on their co-administration with antioxidants.D-methionine and melatonin were shown to reduce theharmful ROS formation caused by the interaction ofAAGs with the cell components (Campbell et al., 2007;Reiter et al., 2011). Melatonin turned out to be particularlyeffective; compared to a mixture of other antioxidants, itwas 150 times more effective in reducing ototoxic effectsof gentamicin or tobramycin.Recently, a new approach to reduce AAG toxicity has

been tested, whereby AAGs were co-administered withsmall molecular mass molecules, derivatives of phthalimide(e.g. CDX5–1) (Baradaran-Heravi et al., 2016). When usedalone, these compounds did not induce PTC-RT, butapplied with AAGs, they efficiently potentiated PTC-RT

stimulating efficacy of AAGs. This strategy allowed signifi-cant lowering of the therapeutic dose of AAGs, thus redu-cing the AAG-related toxicity. For example, CDX5–1administered together with G418 not only increased thelevel of PTC-RT up to 180-fold, but also accelerated syn-thesis of the full-length protein (the full-length product wasdetected 10 times faster). CDX5–1 action did not rely onstimulating PTC-RT; nor did it inhibit the NMD process(Baradaran-Heravi et al., 2016). The mechanism by whichthis compound potentiates PTC-RT activity of AAGs, aswell as its potential interference with other cellular pro-cesses, remains to be elucidated.Other approaches aiming to reduce AAG toxicity related

to their positive electrostatic charge involve the use ofliposome-encapsulated AAGs. Liposomes containing en-capsulated gentamicin, used to stimulate PTC-RT in DMDmice, were more effective and caused 10-fold lower ototox-icity than the traditionally administered gentamicin. Cre-atinine concentrations (an indicator of renal malfunction)in mice treated with these compounds were normal, sug-gesting that encapsulation of AAG also reduced nephrotox-icity (Yukihara et al., 2011; Schiffelers et al., 2001).

Chemical modification of AAGs - AAG derivativesA different approach to the therapeutic use of PTC-RTinvolves modification of the chemical structure of AAGswith a proven nonsense suppressing ability, to obtaincompounds with a reduced toxicity and retained or in-creased therapeutic potential. An example of this ap-proach comes from the studies on artificially designedparomomycin derivatives, where NB30 represents thefirst, and NB54 – the second generation. The modificationof the paromomycin structure effectively reduced toxicityof the derivative compounds. For example, NB30 was 15-fold less toxic than gentamicin, while both compoundsshowed nonsense suppression at the levels similar to thoseinduced by gentamicin and paromomycin (Nudelman et al.,2009). The compounds were able to induce statistically sig-nificant PTC-RT levels in cell lines from patients with dif-ferent PTC-caused diseases, such as: Rett syndrome(Brendel et al., 2011; Vecsler et al., 2011), cystic fibrosis(Rowe et al., 2011), mucopolysaccharidosis type I – Hurlersyndrome (MPS I-H) (Wang et al., 2012; Kamei et al.,2013) and Usher syndrome type 1 (Rebibo-Sabbah etal., 2007; Goldmann et al., 2012).Modification of the structure of another AAG, G418,

resulted in the third generation of PTC-RT-stimulatingAAG derivatives, NB74 and NB84. These compoundswere several-fold more active than the previous genera-tions (Nudelman et al., 2010). Importantly, modificationsalso highly reduced the toxicity of the compounds. Thecapacity of NB74 and NB84 to stimulate PTC-RT wasanalyzed in the mouse model of MPS I-H caused by aPTC mutation in the α-L-iduronidase gene. The level of

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PTC-RT induced by NB84 was higher than that causedby gentamicin or by the first and second generation ofAAG derivatives, and the LC50 values were 9–10 timeshigher than for G418 (Shulman et al., 2014). A long-term (28-weeks) treatment of MPS I-H mice with NB84resulted in an increased activity of α-L-iduronidase indifferent tissues, including the brain, heart and bone.The level of the enzyme was sufficient to significantly re-duce disease symptoms in the tissues, while no visibletoxicity was observed; moreover both the NB84 andNB74 compounds were shown to be able to cross theblood-brain barrier (Gunn et al., 2014). The newest rep-resentative of the third generation of chemically modi-fied AAGs, NB124, turned out to be even morepromising. Its therapeutic index (comparison of theamount of a compound that causes the therapeutic ef-fect to the amount that causes toxicity) was 10 timesbetter than that obtained for gentamicin and other non-modified AAGs. Moreover, a series of cell and animalbased experiments using the CF models have shown thatNB124 restored up to 10% of the wild-type CFTR func-tion (Xue et al., 2014). In the most recent study, usingreporter vectors in the transiently transfected cell lines,NB124 was shown to efficiently decode common PTCmutations in tumor suppressor genes (p53 and APC) inthe murine cell line (NIH3T3), resulting in over 20% ofthe PTC-RT. In the HDQ-P1 cell line (derived from hu-man primary breast carcinoma), the restoration of p53and APC tumor suppressor proteins expression byNB124 induced apoptosis in 38% of the cells, whereasuntreated cells displayed no signs of apoptosis. Inaddition, similarly to G418, NB124 also affected theNMD process, inhibiting the degradation of p53 tran-scripts (Bidou et al., 2017).Another modified AAG is a pyranmycin (TC007), a

derivative of another aminoglycoside antibiotic, neomy-cin (Chang et al., 2002). In the fibroblasts from patientswith spinal muscular atrophy (SMA) caused by the pre-mature termination of the SMN protein, stimulationwith TC007 resulted in the 10-fold increase in the levelof full-length SMN protein. In a mouse model of SMA,injection of TC007 into the central nervous system(brain ventricles) induced higher expression SMN pro-tein, led to a longer survival of the motor neurons andwas associated with a 27% increase in the lifespan of theSMA mice (Mattis et al., 2009; Mattis et al., 2012).

Readthrough compoundsApart from modification of AAG structure, a numberof studies have searched for readthrough-inducingcompounds, chemically not related to AAG, but pos-sessing the potential to suppress nonsense mutations inmammalian cells (Lee & Dougherty, 2012). The best

known example of this group is a small moleculedrug 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]-benzoicacid, also known as PTC124, Ataluren or Translarna. Identi-fied during a high-throughput screen, PTC124 has beenselected as the most potent readthrough promoting drugfrom over 800,000 compounds screened (Welch et al.,2007).PTC-RT stimulating efficiency of PTC124 was success-

fully demonstrated in animals (Goldmann et al., 2012;Welch et al., 2007). It was also tested in different in vitroand ex vivo models of PTC-mediated diseases, includingcystic fibrosis (Du et al., 2008), Duchenne musculardystrophy (Yukihara et al., 2011; Finkel et al., 2013),Miyoshi myopathy (Wang et al., 2010), Usher syndrome(Goldmann et al., 2012) and Batten disease (Sarkar et al.,2011). Phase I and phase II clinical trials have shown thatits adverse effects were mild or moderate and similar tothe placebo-treated group; therefore, PTC124 has beendeemed safe for therapeutic use in humans (Hirawat et al.,2007; Kerem et al., 2008), and a number of clinical trialshave been undertaken. Unfortunately, despite many posi-tive results from different experimental models, the effectsof PTC124 in patients remain inconclusive.In CF patients with PTC mutations in the CFTR gene,

treatment with PTC124 slightly increased chloride chan-nel activity, resulting in an improvement of clinical pa-rameters (Kerem et al., 2008); however, not all patientsresponded positively to the drug. In a similar clinicaltrial with pediatric CF patients, an improvement inchloride channel activity was observed again, and thepresence of CFTR protein in nasal epithelium was con-firmed by immunofluorescence; nevertheless, no signifi-cant therapeutic improvement was proved (Sermet-Gaudelus et al., 2010). In the phase III clinical trials,some improvement in lung function and reduction inexacerbation frequency were reported after 48 weeks ofPTC124 therapy, although statistical significance in theoverall patient population was not reached (Kerem et al.,2014). The main outcome of another phase III clinicalstudy (ClinicalTrials.gov; id: NCT02139306, finished inMarch 2017), has been announced via the PTC Thera-peutics press release, and at the time of writing thisreview, the full data were still unpublished. Theannouncement reported a failure to reach the expectedprimary and secondary endpoints. In light of thesediscouraging results, PTC Therapeutics has recentlydecided to discontinue their clinical development ofPTC124 for CF and withdraw its application formarketing authorization in Europe (PR Newswire).PTC124-induced suppression of PTC was also tested in

clinical trials with DMD patients. In the phase IIA trial, anincreased expression of the full-length dystrophin wasshown in patients’ biopsies after 28 days of oral adminis-tration of PTC124; one third of the patients demonstrated

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an increase in the post-treatment expression of dystrophinappropriately localized in the sarcolemmal membrane ofmuscle cells (Finkel et al., 2013). In the phase IIb trial, thepatients showed a slower decline in the walk test; no im-provement related to the higher dose of PTC124 was ob-served, suggesting a bell-shaped dose-response curve(Bushby et al., 2014). Unfortunately, phase III clinical studycompleted in 2017, indicated that similarly to the CF studies,the effects of PTC124 treatment did not differ significantlyfrom the placebo group (McDonald et al., 2017).Despite the intensive studies, the exact mechanism of

PTC124 action remains unknown. Computational model-ing suggested that mRNAs containing PTC form stablecomplexes with PTC124, which may interfere with therecognition of PTC by eRF1 and suppress the terminationof translation; however, this interaction was shown to bestable only for the UGA codon (Lentini et al., 2014). Incell lines transfected with different PTC-bearing con-structs, the PTC-RT-stimulating potential of PTC124 wasdifferent for distinct stop codons; UGA suppression wasthree times more effective than UAG, and 6 times moreeffective than suppression of UAA (Welch et al., 2007).The latest evidence indicates, that PTC124 has a selectiv-ity for the ribosomal A site and that it promotes insertionof nc-tRNAs at the PTC site (Roy et al., 2016). Tobra-mycin, a compound with an affinity for the ribosomal Asite, is a strong inhibitor of PTC124 (probably by competi-tion), what supports the expected interaction of PTC124with the ribosome (Roy et al., 2016; Salian et al., 2012).Moreover, in a post-hoc analysis of the recent clinical trial,it was observed that CF patients with nonsense mutationsfailed to show a significant response to administeredPTC124, if they were concurrently treated with the in-haled tobramycin (Kerem et al., 2014).Clitocine [6-amino-5-nitro-4-(β-D-ribofuranosylamino)

pyrimidine] has been identified in the same high-throughput screen as PTC124 (Welch et al., 2000). Thiscompound is an adenosine nucleoside analog origin-ally isolated from the mushroom Clitocybe inverse(Kubo et al., 1986). Clitocine is incorporated into mRNAduring transcription as an adenosine substitute; unlikeAAGs that act on the translation machinery, clitocine fa-cilitates decoding of the nonsense codon just by its pres-ence in the transcript (Friesen et al., 2017). Clitocine canbe incorporated instead of the adenine in each of thestop codons. The order of readthrough susceptibilityof the stop codons in the presence of clitocine isUAA> >UGA >UAG. Surprisingly, the UAA codon is themost leaky, probably because of the incorporation of clito-cine in both the second and third position of the codon(Friesen et al., 2017). Stop codons with the incorporatedclitocine are poorly recognized by the eRF1 translationtermination factor; this prevents efficient termination, en-hances nc-tRNA recruitment and therefore increases the

level of PTC-RT. In the mentioned studies, a full-lengh p53protein that was translated due to the readthrough was fullyfunctional, both in the cell lines with PTC-containing allelesand in the tumor-bearing mice (Friesen et al., 2017).Apart from PTC124, a number of other low-molecular-

mass compounds, capable of stimulating PTC-RT, havebeen identified to date. Screening of ~ 34,000 compoundsusing a novel high-throughput screening assay identifiedtwelve most promising readthrough-stimulating com-pounds (Du et al., 2009b). The compounds displayedreduced toxicity in the mammalian cells and did not alterthe global protein expression patterns (Gatti, 2012). Twoleading first generation compounds, RTC13 and RTC14,were proven to suppress nonsense mutations in ataxia tel-angiectasia patients’ cell lines (A-T cells) and in myotubecells from mdx mice (Du et al., 2009b; Kayali et al., 2012).More recent studies identified the further PTC-RT-stimu-lating compounds, namely GJ071 and GJ072, their an-alogs (RTC204 and RTC219), as well as non-relatedcompounds, BZ6 and BZ16 (Jung et al., 2011; Du etal., 2013). In the A-T cells, these compounds displayedPTC-RT-stimulating efficiency comparable to RTC13,RTC14 and PTC124, but with significantly lower toxicity.When tested in cell lines from patients with four differentlysosomal storage diseases, RTC13, RTC14, BZ6 and BZ16showed ~ 1.5 fold increase in the amount of a relevantfull-length mRNA compared to the previously used com-pounds (Gómez-Grau et al., 2015) and were able to sup-press all three stop codons (Du et al., 2013). However, themechanism of their action remains unknown.New readthrough compounds have also been found

among non-AAG antibiotics. A dipeptide antibiotic,negamycin, structurally not related to AAG, affects theprocess of ribosomal decoding in a way similar to AAGs(Arakawa et al., 2003). However, in contrast to AAG, nega-mycin exhibits much lower cytotoxicity and no ototoxicity.Negamycin was shown to promote PTC-RT in the dys-trophin gene both in vivo in the mouse model of DMD andin the cultured mdx myotubes (Arakawa et al., 2003). Itwas also tested in congenital muscular dystrophy: PTC-RTinduced by negamycin in the transfected NIH3T3cells was several-fold higher than that induced by AAGs(Allamand et al., 2008).Another group of compounds capable of inducing

PTC-RT are macrolides, antibiotics often used to treatGram-positive bacterial infections. Induction of PTC-RT by tylosin, josamycin, and spiramycin was demon-strated in colorectal cancer models (mutations in the APCgene) (Zilberberg et al., 2010). The effect was also con-firmed in a xenograft mouse model – treatment withmacrolides resulted in reduced tumor growth and led to anotable increase in the lifespan of the mice (Zilberberg etal., 2010). These data were later extended by the flowcytometry-based reporter assay, in which erythromycin

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and its derivative, azithromycin, were used (Caspi et al.,2015). Macrolides were analyzed in cell lines derived frompatients with mutations in a number of genes; ATMin ataxia-telangiectasia (A-T), MeCP2 in RTT syndrome,SMN in the SMA syndrome, APC in familial adenomatouspolyposis (FAP). In all these cases, macrolides inducedPTC-RT; additionally, azithromycin worked at a 100-foldlower concentration than any other compound tested sofar, enabling a significant reduction of the therapeutic dose(Caspi et al., 2015). PTC-RT-stimulating potential ofazithromycin was also confirmed in SMA mouse modelafter intracerebroventricular administration of this drug(Osman & Iii, 2017). The mechanism of macrolide actionin PTC-RT differs from that of AAGs: according to stud-ies on bacteria, macrolides are thought to influence themechanism of protein synthesis termination, either by re-ducing binding of the RF1 or RF2 to the A-site of the ribo-some, or by inhibiting the release of a peptide from theribosome after binding of the release factors is completed(Thompson et al., 2004).Identification of novel readthrough compounds among

antibiotics has inspired the search for readthrough-stimulating properties among other clinically approveddrugs, which already have an existing safety profile. Ascreen of 1600 clinically approved drugs identified anherbal anti-inflammatory drug, escin (Mutyam et al., 2016).Escin induced a significant PTC-RT, both in the in vitrotests and in the cell lines from a CF patient. It also stabi-lized transcripts by inhibiting NMD, which resulted in asignificant increase of the functional protein level in thecells. No side effects have been reported so far, and escinseems to be a promising PTC-RT-stimulating compound.However, further functional tests need to be performed be-fore it can be used in clinical trials.

Readthrough induced by the use of suppressor tRNAsThere is an alternative for the stop suppression stimulatedby readthrough compounds - the suppressor tRNAs.Suppressor tRNAs are altered aminoacylated-tRNAs withanticodons complementary to stop codons (Beier &Grimm, 2001). They do not stimulate translational ma-chinery like others readthrough compounds, but they out-compete translation termination factors (like nc-tRNAs),and drive incorporation of an amino acid, being thereforereadthrough agents per se. Suppressor tRNAs have beenshown to restore the expression of functional proteins inPTC-carrying human cell models of beta-thalassemia(Temple et al., 1982), xeroderma pigmentosum (Panchalet al., 1999), Ulrich disease (Sako et al., 2006) and, morerecently, cancer (Bordeira-Carriço et al., 2014). The PTCsuppression using suppressor tRNA was also shown inmice, albeit with very low efficiency (Buvoli et al., 2000).In contrast to the readthrough compounds, which stimu-late PTC recognition by nc-tRNAs (resulting in the

incorporation of amino acid that does not necessarily cor-respond to the wild-type), suppressor tRNAs do not intro-duce missense mutations, and thus lead to the synthesis ofthe functional protein (Bordeira-Carriço et al., 2014). How-ever, a clinical use of suppressor tRNAs faces many chal-lenges, especially regarding the efficiency of delivery and invivo expression of these systems, with minimal toxicity tothe organism.

Bioavailability of the drugs used to stimulate PTC-RTWhen drug-stimulated PTC-RT is considered in thecontext of its possible therapeutic application, bioavail-ability of the readthrough compounds, both at the levelof the cell as that of the organism, is an important issue.

The cell membrane permeability to readthroughcompoundsPTC-RT may be limited by the permeability of the cells toreadthrough compounds. Uptake of the drugs, especially ofAAGs, has been explored for many years, but still no un-equivocal conclusions have been reached. Even less isknown about the mechanisms of the cell penetrance by theAAG derivatives or non-AAG compounds with PTC-RT-stimulating potential.Nearly all mammalian cells take up AAGs; these drugs

may cross cellular membranes via endocytosis or non-endocytotic pathways (Steyger, 2005). One of the beststudied AAGs uptake mechanism is through the multi-ligand endocytic receptor – megalin. Megalin has beenlocalized in the apical membrane of the epithelial cells ofthe proximal renal tubules and in the hair cells of the innerear, and is related to the nephro- and ototoxic effects ofAAGs (Tauris et al., 2009; Christensen et al., 2012). How-ever, at low temperature, when endocytosis is significantlyslowed down, the presence of Texas Red-tagged gentami-cin (GTTR) in the cytoplasm or the gentamicin-relatedROS production occurred within seconds (Myrdal et al.,2005; Hirose et al., 1997). This suggests that AAGs uptakeinvolves different mechanisms than endocytosis alone.A potential non-endocytic route of AAGs uptake is

through non-selective cation channels (NSCCs), such asTRP channels (Marcotti et al., 2005; Lee et al., 2013;Stepanyan et al., 2011). Low Ca2+ level in the extracellularfluid, hyperpolarization of the cell membrane or applica-tion of NTSCC regulators (e.g. TRP agonists: resinifera-toxin and anandamide) activate NSCCs opening, induceinflux of cations along with the influx of positively chargedcompounds. Factors inducing activation of NSCCs open-ing also favor AAGs uptake (Steyger, 2005). Similar tomegalin, TRP channels are present in the membrane ofkidney epithelial cells and of inner ear sensory cells, thusmay also be involved in oto- and nephrotoxic effects ofAAGs. Moreover, AAGs uptake may also be stimulated byloop diuretics, like bumetanide and furosemide. These

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drugs, which hyperpolarize the cell membrane, wereshown to increase the influx of a cationic AAG (GTTR)through NSCCs, enhancing GTTR uptake by 60% (Wanget al., 2013).

Effective dose of readthrough compoundsRegarding the modes of administration, the choice ofdoses and duration of therapy is important. The stimula-tion of PTC-RT is a transient phenomenon, and thetherapeutic use of drugs requires a repetitive, prolonged,usually life-long administration of the compounds. Moststudies in animal models indicate that the action ofAAGs follows a peak-driven mode. For example, in themdx mice, maximal levels of intravenously injected genta-micin in serum were observed 20 min after administra-tion; after 4 h, the drug level rapidly decreased. They alsoshowed that a constant pump-driven administration oflow-concentrations of gentamicin did not result in read-through (Barton-Davis et al., 1999). It was confirmed inretinitis pigmentosa mice, that a better response to thetreatment was obtained with repeated single injections ofa higher dose than with continuous delivery of small dosesof AAGs (Guerin et al., 2008). In the clinical trials with CFpatients, different regimens of the administered gentami-cin were used (Clancy et al., 2001; Sermet-Gaudelus et al.,2007; Malik et al., 2010). In the Clancy’s study, the schemeof intravenous administration of gentamicin (2.5 mg/kgthree times a day) was adjusted to achieve peak concentra-tion in the serum from 8 to 10 μg/ml (Clancy et al., 2001).In Sermet-Gaudelus study, gentamicin was adminis-tered at 10 mg/kg once daily for 15 days; gentamicinpeak concentration was achieved 30 min after the in-fusion (Sermet-Gaudelus et al., 2007). In another longterm study, gentamicin was administered intraven-ously (7.5 mg/kg) once or twice per week for6 months; gentamicin efficacy was limited and its tox-icity precluded administration of higher doses of thisdrug (Malik et al., 2010).Effective dose of the best studied non-aminoglycoside

compound, PTC124 was shown on healthy volunteersand CF patients in phase I and II clinical trials. PTC124was characterized by rapid oral absorption and dose-proportional increases in pharmacokinetic parameters.Peak concentration of this compound was achieved atapproximately 2 h after dosing and its half-life rangedbetween 3 to 6 h (Hirawat et al., 2007). Similarly toAAGs, the best response for the treatment was observedafter a multi-dose administration, however without toxicdrug accumulation or metabolic auto-induction. The dos-age used in clinical trials varied from 16 mg/kg/day in a14-day treatment period for the first studies to the 40 mg/kg/day in three divided doses for 48 weeks (Kerem et al.,2008; Sermet-Gaudelus et al., 2010). Generally, choosing

the most effective regimen of drug administration for agiven disease remains challenging.

Other factors influencing therapeutic potential ofdrug-induced PTC-RTThe efficiency of PTC-RT stimulation, the toxicity and thebioavailability of stimulating compounds are important is-sues to be addressed. As presented above, these problemsare potentially manageable, mainly through the search fornew, more efficient and less toxic compounds, for themeans to enhance cellular uptake of the drugs and for theefficient ways to deliver these drug to proper tissues.Other PTC-RT-related problems are even more elusive

and finding ways to overcome them may be difficult, ifpossible at all.The availability of a PTC-bearing transcript, which can

affect the efficiency of the nonsense suppression, is onesuch issue. In the cell, mRNA availability is strictly re-lated to the efficiency of the NMD process (Miller &Pearce, 2014). When NMD is efficient, the level of mu-tant mRNA is noticeably reduced and, even if potentnonsense suppressing drugs are provided, the amount offunctional protein is very low (Kuzmiak & Maquat,2006). In the study using gentamicin as the PTC-RT-stimulating drug in CF patients carrying the sameW1282X nonsense mutation, the response to gentamicinwas enhanced in the individuals, in whom the level ofmutant mRNA was high due to low NMD efficiency(Linde et al., 2007). These results suggest that the levelof PTC-bearing transcripts might be a limiting factor inthe response to gentamicin treatment. Some reports sug-gested that even a moderate induction of PTC-RT (e.g.by using AAGs) may promote a stabilization of mutanttranscripts and counteract the NMD process (Bedwell etal., 1997). This hypothesis has been later supported byseveral reports (Floquet et al., 2011; Salvatori et al.,2009; Bellais et al., 2009).Since the level of mutated transcript expression is of a

paramount importance for PTC-RT therapy, blocking NMDby pharmaceutical agents has been considered as the solu-tion to the problem, and a number of studies have exploredthis path (Usuki et al., 2004; Usuki et al., 2006; Durand et al.,2007; Keeling et al., 2013; Gotham et al., 2016).Inhibitors of hSMG-1 kinase, which is an essential

protein for the regulation of NMD process, form one ofthe groups of tested compounds (represented by wort-mannin and caffeine) (Usuki et al., 2004). In the experi-ments performed in fibroblasts derived from patientswith Ullrich’s disease, caused by PTC in the collagen VIgene, the use of these agents resulted in a correct assem-bly of collagen VI, despite its truncated C-terminus andformation of a partially functional extracellular matrix(Usuki et al., 2004; Usuki et al., 2006). Another molecule,a small polycyclic indole derivative, NMDI-1, was shown

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to block the NMD process by interfering with theinteraction between key NMD factors, hSMG5 andhUPF1. This led to the stabilization of the hyperpho-sphorylated form of hUPF1 and its sequestering incytoplasmic P-bodies (Durand et al., 2007). NMDI-1was shown to be ~ 1500-fold more effective than caf-feine in attenuating NMD; at the same time, it didnot have any influence on cell growth and no signifi-cant effect on protein synthesis (Keeling et al., 2013).Short term studies in a mouse model of mucopolysacchar-idosis (MPS I-H) have shown that a co-administration ofNMDI-1 with gentamicin restored 50% more α-L-iduro-nidase activity than AAG administered alone (Keelinget al., 2013). Chemical synthesis of NMDI-1 is compli-cated and poorly efficient; however, there were made at-tempts to synthesize a close NMDI-1 analog, VG1, withsimilar NMD inhibiting potential and significantly lesscomplicated synthesis protocol (only 6 synthesis steps in-stead of 13 reported previously) (Keeling et al., 2013;Gotham et al., 2016).Another promising NMD inhibitor, amlexanox, is a

long-used drug with anti-allergic and anti-inflammatoryproperties (Makino et al., 1987). Amlexanox has beenapproved by the FDA for the treatment of canker sores,aphthous ulcers and asthma; it is also currently in aphase II clinical trial for the treatment of diabetes(ClinicalTrials.gov; id: NCT01842282). The recent studiesshowed that amlexanox can inhibit NMD process andpromote synthesis of PTC-bearing mRNAs (Gonzalez-Hilarion et al., 2012). The increase in the amount ofPTC-bearing mRNA does not affect general transla-tion or expression of potential NMD substrates (Gon-zalez-Hilarion et al., 2012). In the cell lines from CFpatients, amlexanox alone caused an increase ofcAMP-dependent halide efflux, suggesting presence ofthe functional CFTR protein, probably due to thesuppression of PTC in the mutated CFTR transcript.The increase in CFTR activity after treatment withamlexanox alone was ~ 3 times higher than aftertreatment with other PTC-RT inducing compounds,G418 or PTC124. Although the initial results forthese agents seem promising, long-term safety andeffectivity studies are required before the clinical useof these compounds becomes possible. Efficacy ofamlexanox as a readthrough compound and a potentNMD inhibitor was confirmed recently in cells de-rived from patients with recessive dystrophic epider-molysis bullosa, where a significant increase in a full-length protein level translated from PTC-bearing tran-scripts was observed (8–80%), compared do the wild-type protein level (Atanasova et al., 2017). The recov-ered protein was also functional and stable.The inherent problem associated with the PTC-RT-

based therapies is related to the fact that the level of the

induced readthrough can vary considerably for differentPTC introducing mutations, such that only a subset ofpatients would be likely to profit from the therapy(Malik et al., 2010; Woodley et al., 2017; Finkel et al., 2013;Nagel-Wolfrum et al., 2016). This variability depends onthe stop codon itself and/or on the sequence context ofPTC (Dabrowski et al., 2015). The mechanism of the in-duced stop codon suppression still remains not fully under-stood, complicating the prediction of PTC-RT efficiencybased on the nucleotide context of the PTC mutation. Tosome extent, this problem may be solved if the efficient pairof the PTC and a specific readthrough compound is found.This however has to be done in an empirical way –to pre-dict which patients are likely to take benefits from thePTC-RT therapy, it is necessary to experimentally deter-mine the PTC-RT level of each nonsense mutation in pre-clinical settings (Floquet et al., 2012). Moreover, one has tokeep in mind that some of the PTCs, in specific sequencecontext, may be resistant to the drug-induced PTC-RT(Bukowy-Bieryllo et al., 2016).Even if the full-length protein is produced after drug-

induced PTC-RT, the important question about theidentity of the amino acid incorporated during decodingof the premature stop still remains. If it is the sameamino acid as that present in the normal protein, thequestion can be disregarded. However, if the amino acidis different from the wild-type, it can lead to synthe-sizing a potentially non-functional protein, with differ-ent biochemical properties or impaired stability. Anumber of studies showed that, even if the full-length pro-teins were produced after administration of PTC-RT-inducing drugs, their functional activity and subcellularlocalization were often impaired (Schulz et al., 2002; Yaoet al., 2009; Simon et al., 2010; Brumm et al., 2012).Some preferences for the amino acid incorporated

during the PTC decoding can be predicted from thenear-cognate anticodons (Dabrowski et al., 2015). How-ever, this is not the unequivocal indicator. In vitro stud-ies in rabbit reticulocyte lysates identified Trp, Arg, andCys incorporated at the UGA codon (Feng et al., 1990).Studies addressing the insertion of amino acids at UAAand UAG codons in viral mRNAs revealed only the pres-ence of Gln (Feng et al., 1990; Yoshinaka et al., 1985). Re-cent readthrough studies using mass-spectrometry shedmore light on this issue (Roy et al., 2015; Roy et al., 2016;Xue et al., 2017). Sequence analysis of full-length read-through products in the yeast system (with reporter vec-tors containing different stop codons) has shown that Gln,Lys, and Tyr incorporated at UAA and UAG, whereasTrp, Arg, and Cys were inserted at UGA. This was irre-spective of whether readthrough is endogenous, or in-duced. However, the frequency of individual amino acidsinsertion differed for specific nonsense codons and read-through compound; Tyr and Gln were incorporated with

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comparable frequency at UAA (~ 45% and ~ 55%, respect-ively), Gln dominated at UAG (~ 80%) and Trp at UGA(~ 86%). Interestingly, for all these amino acids, the nc-tRNAs mispaired at position 1 or 3 of nonsense codons.Slightly different results were obtained a human cell linetransfected with reporter vectors containing different stopcodons (Roy et al., 2016) (Table 1). The frequencies of theincorporated amino acids were different and mispairing atposition 2 of nonsense codons was allowed. Nevertheless,the set of possible amino acids incorporated at PTCs wassimilar to that in the yeast system. The newest evidenceindicates that, after stimulation with G418, identity ofinserted amino acids depends on the nucleotide context ofthe same stop codon (UGA). This confirms, that mRNAsequence context plays a key role in near-cognate tRNAselection during PTC-RT (Beznosková et al., 2016; Xue etal., 2017).An important issue in PTC-RT based therapies is the

minimal amount of the full-length protein that shouldbe restored to achieve the functional and – hopefully –therapeutic effects. In each disease, this level is different:in CF, 30–35% of normal CFTR activity is sufficient; inDMD – 20-30% of the full-length dystrophin (but ac-cording to others, even 1% is sufficient); in MPS I-H,even 0.4–1% of the α-L-iduronidase level is enough toalleviate disease symptoms (Hoffman et al., 1988; Keel-ing et al., 2014). Unfortunately, the effective amount ofthe full-length protein has to be examined for each geneand disease separately.

ConclusionsTo date, more than 100 studies have shown that thestimulation of PTC-RT may result in the partial res-toration of the expression of deficient proteins thatunderlie ~ 40 different genetic diseases (Lee & Dougherty,2012; Nagel-Wolfrum et al., 2016). However, no study hasdemonstrated a remarkable improvement of patients’

health or significant alleviation of the disease phenotype.The establishment of a truly efficient PTC-RT-basedtherapy requires new, more potent compounds withless toxicity. It can be achieved by the modification ofavailable drugs or by searching for completely newcompounds with higher nonsense suppression poten-tial. Additional enhancement might be ensured by co-administration of readthrough compounds with someenhancers, like inhibitors of NMD process or amino-glycoside potentiators, such as CDX5–1. Despite thepromising results of the basic research and clinicaltrials, plenty of work is still needed to better under-stand/predict ADME (absorption, distribution, metab-olism and excretion) characteristics, pharmacokinetics(PK) and pharmacodynamics (PD) of a drug, beforethese new drugs can be considered as a clinical thera-peutic option (Lee & Dougherty, 2012).

AbbreviationsAAGs: Aminoglycoside antibiotics; eRF1/3: Eukaryotic release factor 1/3;GTTR: Texas Red-tagged gentamicin; N c-tRNA: Near-cognate tRNA;NMD: Nonsense-mediated mRNA decay; NTC: Normal termination codon;PAA: Poly-L-aspartic acid; PABP: Poly(A)-binding proteins; PTC: Prematuretermination codons; PTC-RT: Premature termination codons readthrough;ROS: Reactive oxygen species; RT: STOP codon readthrough

FundingThis work was supported by the National Science Centre, Poland; grantsno. 2013/09/D/NZ4/01692 (ZBB); 2016/20/T/NZ4/00525 (MD) and2016/23/N/NZ4/03228 (MD).

Authors’ contributionsMD - a major contributor in writing of the manuscript, ZBB - editorial andsubstantive corrections, EZ - manuscript supervisor. All authors read andapproved the final manuscript.

Ethics approval and consent to participateNot applicable

Competing interestsThe authors declare that they have no competing interests.

Table 1 Amino acids inserted during readthrough

Basal PTC-RT

UGA UAA UAG

Arg (CGA)(58.6 ± 2.3%)

Trp (UGG)(41.4 ± 2.3%)

Cys (UGU/C)-

Tyr (UAU/C)(67.5 ± 8.9%)

Gln (CAA)(32.4 ± 8.9%)

Tyr (UAU/C)(73.1 + 15%)

Gln (CAG)(25.2 ± 13.7%)

Trp (UGG)(1.3 ± 1.4%)

Stimulated PTC-RT PTC124 (30 uM)

UGA UAA UAG

Arg (CGA)(69.7 ± 11.3%)

Trp (UGG)(28.8 ± 11.4%)

Cys (UGU/C)(0.7 ± 0.7%)

Tyr (UAU/C)(57.9 ± 11.3%)

Gln (CAA)(39.9 ± 11.7%)

Tyr (UGG)(43.9 ± 20.9%)

Gln (CAG)(53.2 ± 20.3%)

Trp (UGG)(1.8 ± 1.9%)

Stimulated PTC-RT G418 (150 uM)

UGA UAA UAG

Arg (CGA)(64.5 ± 11.8%)

Trp (UGG)(17.9 + 6.8%)

Cys (UGU/C)(17.7 ± 8.0%)

Tyr (UGG)(47.9 ± 14.1%)

Gln (CAA)(52 ± 14.2%)

Tyr (UAU/C)(10.8 ± 7.0%)

Gln (CAG)(86.5 ± 8.3%)

Lys (AAG)(2.0 ± 0.8%)

The frequency of amino acids inserted during either basal or stimulated readthrough (PTC124 or G418) in the human cell line transfected with reported vectorscontaining different stop codons (based on Roy et al., 2016)

Dabrowski et al. Molecular Medicine (2018) 24:25 Page 11 of 15

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in publishedmaps and institutional affiliations.

Received: 6 April 2018 Accepted: 1 May 2018

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